The goals of this application are to apply tools and refine methodologies for genome engineering in zebrafish that allow the creation of mutagenic and conditional alleles that provide spatial and temporal control of gene expression. In doing so we will be able to define genes that aid in the development and implementation of methodology to improve our understanding of genetic manipulations that can promote or restore health. The ability to make site-specific, untagged mutant alleles in zebrafish and other models has been greatly advanced by custom nucleases that include TALENs and CRISPR/Cas9 systems. These systems operate on the same principle: they are designed to bind to specific sequences in the genome and create a double strand break. During the first granting cycle, we have leveraged this ability to develop reliable methods for site-specific gene integration directed by short regions of homologous sequence. In our renewal application, we utilize the tools, vectors and methodologies generated to create both revertible alleles and Cre/Cre-ER lines for the zebrafish community in Aim 1. In Aim 2, we will examine methodologies to improve site-specific integration and the role of microhomology at CRISPR/Cas9 cut sites and new ways to present the vector with microhomology to the genomic cut site. In Aim 3, we will continue to develop and implement targeted integration resources by hosting workshops and onsite visits at both the Mayo Clinic and Iowa State University. The tools, techniques and zebrafish lines produced will have direct implications for providing precise gene editing techniques and critical lines to examine genes with the potential to restore human health. We anticipate these methodologies will enhance the efficiency of gene editing and will be readily adaptable for use in other model organisms and large animals. In our opinion, this will have important implications for modeling human disease in animal systems through the ability to utilize conditional alleles. The methods, cargos and zebrafish lines are designed to significantly enhance identification of genes that promote health through leveraging genome modification to produce conditional and revertible alleles and alleles that mirror mutations in humans.